There is a fundamental need for novel transformative approaches to dissecting important biological processes at a system, multi-gene, level rather than one gene at a time. Single gene approaches are often flawed by the complex feed-back and feed-forward mechanisms as well as redundancies involved in biological systems. Without more comprehensive knowledge of all the pathways involved in a particular biological process, it will be exceedingly difficult for biologists and clinicians to manipulate these processes to improve human health. The long-term goal of the lab is to use the small non-coding RNAs, miRNAs, to provide a more complete map of all the pathways involved in specific biological outcomes. The objective here is to use miRNAs to dissect most, if not all, the pathways required to promote the dedifferentiation of adult somatic cells to induced pluripotent stem cells. The central hypothesis is that one can use the unique features of miRNAs, which have multiple targets with common physiological outcomes, as a robust means to uncover proteins, pathways, modules within pathways, and cellular processes underlying the reprogramming to induced pluripotency. This hypothesis derives from preliminary data showing how specific miRNAs can influence reprogramming and that, while these miRNAs have hundreds of targets each, the targets can be organized into pathways and protein networks that provide an increasingly comprehensive knowledge of the mechanisms of reprogramming. The following specific aims are proposed: 1) Improve miRNA target predictions based on network associations, 2) Use predictions to dissect all pathways by which a single family of miRNAs promotes self-renewal and pluripotency, 3) Determine most, if not all pathways, that regulate reprogramming through a genome-wide miRNA approach. In Aim 1, a combination of molecular experiments and empirically tested association filters will be used to define network based parameters that more accurately and comprehensively identify targets of individual miRNAs. In Aim 2, molecularly and bioinformatically identified targets of the ESCC miRNAs will be individually tested for their influence on reprogramming, cell cycle, and self-renewal as will the pathways to which the targets are associated. In Aim 3, all miRNAs will be tested for their influence on reprogramming, their targets organized into networks based on positive versus negative influences, and resulting enriched networks tested experimentally. This proposal is highly significant as it provides novel paradigms for uncovering molecular mechanisms underlying physiological processes. While focused on reprogramming, the tools and approach developed by the described experiments could be used to help systematically dissect any process of interest. Such systems level knowledge will allow for more intelligent manipulation of a process to reach a desired outcome required for the better treatment of disease. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because it develops novel means of dissecting the multiple molecular pathways involved in a biological process, which will enable more comprehensive and intelligent means of manipulating cells in the treatment of anything from degenerative disease to cancer. Furthermore, the initial focus is on induced pluripotency, which has the potential to produce replacement tissues as well as provide new and powerful tools to study human disease. Therefore the research is relevant to NIH's mission to foster fundamental creative discoveries that increase the Nation's capacity to protect and improve human health.